Download Tobacco (Nicotiana tabacum L.)-A model system for tissue culture

Indian Journal of Biotechnology
Vol 3, April 2004, pp 171-184
Tobacco (Nicotiana tabacum L.)-A model system for tissue culture
interventions and genetic engineering
T R Ganapathi', P Suprasanna', P S Rao' and V A Bapat'?
IPlant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology
Division
Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India
lndo-American
2
Hybrid Seeds (India) Pvt Ltd, Bangalore 560 070, India
Tobacco (Nicotiana tabacum L.) has become a model system for tissue culture and genetic engineering over the past
several decades and continues to remain the 'Cinderella of Plant Biotechnology', An ill vitro culture medium (Murashige
and Skoog, 1962), based on the studies with tobacco tissue cultures, has now been widely used as culture medium
formulation for hundreds of plant species. Studies with tobacco tissue culture have shed light on the control of ill vitro
growth and differentiation. Further, induction of haploids, microspore derived embryos and selection of mutant cell lines,
have been achieved successfully. Tobacco has also been employed for the culture and fusion of plant protoplasts, providing
invaluable information on way to explore the potential of somatic hybridization in other crops. Optimization of genetic
transformation, using Agrobacterium tumefaciens and A. rhizogenes, has been central to the cascade of advances in the area
of transgenic plants. Developments in the field of molecular farming for the expression and/or production of recombinant
proteins, vaccines and antibodies are gaining significance for industrial use and human healthcare.
Keywords:
genetic transformation,
proteins, tobacco
molecular farming, plant biotechnology,
IPC Code:
lnt. CI.7 A 01 H 4/00, 5/00, A 61 K 35174,35176,39/002,39/02,39/12;
15/09
Introduction
Advances in plant biotechnology have made a
significant impact in the area of in vitro culture,
genetic manipulation
and newer approaches in
experimental plant biology!". Tn the past 2-3 decades,
plant tissue culture has undergone an exciting
development, providing knowledge about totipotency,
differentiation, cell division, cell nutrition, metabolism, radiobiology, mutations and cell preservation.
While microbial systems have been successfully used
for the production of antibiotics and other useful
compounds, it is now becoming possible to use plant
tissue culture system to produce a wide range of
biologically
active compounds,
like alkaloids,
steroids, phenolics,
vitamins and other useful
chemicals. Further, cell culture could be used for the
purposes of specific biotransformation of organic
molecules. Other more sophisticated approaches of
tissue culture include in vitro selection, protoplast
* Author for correspondence:
Tel: 91-22-25593276; Fax: 91-22-25505151
E-mail: [email protected],n
plant cell and tissue culture, recombinant
C 12 N 15/00, 15/01, 15/05, 15/08,
fusion and plant genetic engineering, which have
shown tremendous potential for application in crop
improvement.
Majority of the discoveries in the field of plant cell,
tissue culture and molecular biology have originated
from the experimentation with tobacco plants 1,7. As a
result, tobacco has become a model system as the
'Cinderella of Plant Biotechnology'. This plant has
been found to be an extremely versatile system for all
aspects of cell and tissue culture research. Working
with tobacco tissue cultures, Murashige and Skoog"
devised an in vitro culture medium that has become
the widely used nutrient formulation for an evergrowing range of plant species. In vitro studies with
tobacco tissue culture, using different physical and
chemical factors, have provided insight into the
control of growth and differentiation. Induction of
haploids and selection of mutant cell lines, owing to
the experiments with tobacco tissue cultures, have
become
useful
tools.
Isolation,
culture
and
regeneration of plants from protoplasts as well as
somatic hybridization, all have been accomplished
using tobacco system, which has provided invaluable
INDI,\N J 1110TITIINOL, i\1'1{1I.
172
information
on
ways
to
explore
the
potential
or
somatic
hybridiz.uion
in other crops.
Tobacco
(Nicotiana /(/!J(fCIIIII
L.; 211=4,,=:18) is a
natural allotetraploid
Iormcd
through
hybridiz.uion
between
two
diploid
(211=2--1) progenitors,
N.
sylvestris
and N. torncntosijonnis
npproxim.ucly
(j
million years ago'), Within J months or time, ;1
tobacco plant goes from seed to next generation seed
and generates
up to a million
up to hundred
seed per plant.
or thousands
the early experiments
to the recent vector
From
DN/\
tobacco
has
research
system.
produced
plunt
remained
;IS the
The
in tobacco.
first
stability
is wry
most
sought
gl'ne
and
or pLUlt genetic
mostly
\\'01'1-: witl:
tobacco.
employed
in
plant
this
is
production
and
on tile
being
or useful
special
Ior
l'nginl:erillg
studies
work
it cannot
tile
compil.uion.
aspects
In
plant
or
described
this
on tobacco
tissue
in which
and gl'nL'lic
is so en0l'l110US
up
therefore,
culture
rcxcurch
tobacco has been
tile W;IY lor improvement
Extensive
ill
In a series
endogenous
study
tile
Skoog
s Iloot
primordia
or an auxin
or
(1/\/\)
adenine
or
promotion
effect
or
or cytokinin
The
the
bud
concentration,
buds
induced
rooting
other
physical
For
tile
instance,
growth
growth
or
temperatures
and
tile
differentiation.
callus
increased
in
the
while
tile
by other components
or N/\/\.
lron:
or tobacco
tobacco
or
have
tobacco
tobacco
callus
such
and
sucrose
diffcrcnti.uion
Skoog':'
(.'i-JJoC)
as
1'1'0111
studied
I Ic observed
with
up to JJoC but 1'01'shoot-bud
tile
011 tobacco
the
til:lt
rise
lonnation,
in
or
single
and
Sussexl(,
cell
(Wisconsin
leading
cultured
J8). Then,
were
subjected
also
been m;lI1Y reports
tissues
W;IS reported
tobacco".
N.
lrom
Bud
to
flower
buds with
to
surgical
the
cultured
Iorm.uion
was
ultra
h.ivc
changes
also
Anther
on
/\ surveyor
Ior
shoots
Similarly,
or growth
I '-.
0 I
production
gellctic
haploid
diploids
conducted on/witl:
the idcnufir.uion
plnnttcts,
followed
have
to anther
or haploids
plant
been
culture
and
evoked
breeders
and
was t Iic recovery
1'01'breeding
and 1'01'cstab-
1'01'mut.uiounl
ion c xpcrimcnts.
studies
;1I1c1
Extcuxi vc studies
anther culture or tobacco Il:1S led to
or criiicn]
ractors
I'm succcssfu]
01' haploids.
Nilsch"2
1'01'the Iirs: time, 1'1'0111
N.
by
have
the role or
has been successfully
among
cell cultures
manipul.u
development
that
'1'1ic major
'I a<.vantage
or homozygous
lishing
about
in
regulators,
tissue cultures
pertaining
technique
interest
,.
gcncucrsts
x
and Pollen Culture
this
tile
considerable
callus
studicd".
on tobacco
literature
that
represents
Irom
S{(OJlC/OIlS
in regenerating
been
eLlla on the effect
and light
of
tobacco
in
(N.
contributed
to the basic knowledge
several Iuciors on differentiation
extended
to other crops.
Studies
cxpl.mts
spot resistance
W;lS transto N. /({b(fC{(IIII~.
Cellular
S{((I\'e/OIlS
structural
temperature
leal'
induced
or a hybrid
in
01' planners
Plants were regenerated
N.
Irom
on organogenesis
Rcgcncr.uion
;1I1d brown
/({/)({C{(III)
tcrrcd
calli.
and
j'Ol'll1 cotyledons
utilized
/\ higher
fuciors,
photoperiod
or a range 01" temperatures
callus
resulted
and
man i IHII:I t ionx.
and
Tile presence
dilfcrcnti.uion,
by 1/\/\
influenced
cultures.
(Kn).
medium
was modified
/)11,
temperatures,
effect
in
particularly
or auxin
cultures.
callus
Kn
shoot
in the medium
level
and a cytokinin
various
sepal primordia
were cultured,
leading
to the
I1L'Ld, xt.uucn
and carpel
primordia
in acropetal
sequence
on tile ;qK'X, Org;ln primordia
or tile flower
been
Nicotiana
with
a systematic
nppronch.
They
demonstrated
tilat dif'Icrcuti.uion
into shoot and root
could be induced
by tile manipulation
or the balance
on
format ion
only
have
in
out
bud
inn ucncc 1-', mak i ng it easy to
l licks
iou.
petiole cultures".
a IIII co-werlHieI' meI ucuon
.
flower
chnngcs
dilfcrcnti.u
experimental
or publications,
1.
.
,.;us 10·10
- reporter I on t I'ic III vitro
in red light.
carried
were
hormonnl
obtained
Cultures
vitro
Oil
in the presence
occurred
induction Irom epidermal cells or tobacco dihnploids,
which were ohtaiucd
from Icrrilc flowers raised [rom
peels or mule sterile pl.uus ':'. Thus, hypohaploid', with
less ih.u: (dihuploidx) chromosomes
or epidermal
cell
culture
were
obtained,
which
were
Ircc
Irom
those
crop systems.
was observed
coni roll i ng
robacco
Studies
studies
parameters
only
and
formation
whereas rooting
in a single
model
a role
ill other
or blue ligill,
There
and
1'01' shoot-bud
regeneration
Shoot-bud
was found to
in tobacco".
l ligl: light intensity
18°C was optimum.
be inhibitory
aniibodicx
medicine
be summed
article,
the
on
proteins,
USl~ ill
.uc
CIIITL'lllly,
In the ;1I\:a or plun: tissue culture
industry,
m.tnipul.uion,
and vast that
paved
recombinant
chemicals
to
gene
out using to\):IC((1, /\11
the rirsl achievements
bused
arc
rcl.ucd
expression
have all been worked
;II'ICI'
pl.uus
Iransgl'nic
the experiments
/\Iso,
trausform.uion,
Scaling
rapid.
with tile uptake or n.ikcd
mediated
gene trnuxfcr,
or acres
200,\
investigations
reported
/({baCIIIII.
011 the
haploid
This was
regeneration
01'
GANAPATHI
et al: TOI3ACCO FOR TISSUE CULTURE INTERVENTIONS
haploid plantlets from anther culture of several
species of Nicotiana. Three promising
lines of
tobacco were raised through anther culture of hybrids
(line MC-161O x Coker 139)23. The new lines exhibited higher resistance to bacterial wilt and black
shank without loosing the agronomic and chemical
features of MC-161O. Similarly, double haploids of
tobacco were raised using anther culture", which
showed high yield, good quality of curved leaves and
disease resistance characters,
achieved in a much
shorter period as compared with the conventional
breeding practice. In China, new varieties of tobacco
(Tangu 1, 2 and 3) have been released using anther
culture by Shangtung Institute of Tobacco and are
being cultivated in about 20,000 acres.
First pollen mitosis was reported to be the critical
stage for Nicotiaua anther culture". The physical
factors,
like light,
temperature
and pH, also
influenced
the pattern of response. For example,
anthers of tobacco kept in the dark at the beginning of
the experiment
responded
better".
The nitrogen
starvation of anther donor plants increased the anther
response and embryo yield'". Chilling of anthers prior
to culture or a cold treatment of flower buds also
enhanced
embryogenesis
in tobacc026.28. Further,
29
Deaton et 0/
studied the vigour and variation
expressed
by pollen plantlets
of tobacco
and
conducted
their
evaluation
trials.
However,
Zeppernick et al30 studied the relationship between
ploidy
level, morphology
and concentration
of
nicotine of haploid and doubled haploids raised
through anther culture.
Depending upon species and other factors, pollen
grains either developed
into embryos or formed
callus, which then differentiated
into embryos upon
transfer to an appropriate medium. Normal embryogenic
process
was usually
observed,
showing
globular, heart and torpedo stages of embryos:".
Haploid plants produced large number of flowers and
these were generally smaller in size. However, in
tobacco anther cultures of Nicotiana, the pollen grains
directly
produced
haploid
plantlets
without
intervention of a callus mass. Moreover, besides the
development of normal embryos, numerous abnormal
embryos in various stages of differentiation
were
detected". Albino plants have also been observed in
tobacco anther cultures32.
In general, success in the production of embryos
from anther cultures depends to a large extent on
various factors such as method of culture, nutritional
173
and
hormonal
composition
of the
medium,
developmental stage of microspore and ontogeny of
pollen embryos as well as the growth condition of the
donor plants. The usefulness of haploids is based on
the assumption that homozygous inbred lines can be
readily and rapidly achieved. Haploids could offer as
supplementary
breeding
lines
for
breeding
programmes and the knowledge gained about culture
method, treatment and growth regulators
can be
applicable for anther culture in other crops.
Studies on Genetic Variation and Mutant Selection
Mutant selection through tissue culture has become
possible due to the occurrence of a high degree of
variability (sornaclonal variation) in cell cultures. The
fact that millions of cells can be cultured in shake
flasks has permitted
the selection
for specific
mutations at cellular level and to regenerate plants
from selected cell lines for specific mutations at
cellular level, and to regenerate field plants", Various
types of chemical and physical mutagens have been
applied to callus, cell suspensions or protoplasts in
attempts to isolate desirable mutants.
Plant cell and tissue cultures have been shown to
cause or allow many genetic changes to take place.
Variability in DNA content and nuclear volume was
noticed in regenerating cultures of tobacco". Increase
in ploidy,
chromosomal
rearrangements,
and
abnormal chromosomes and altered DNA content in
regenerating plantletshave
been effectively used in
tobacco improvement
programmes.
The fate and
possible role of aberrations (dicentric chromosomes)
were studied on the development
of the different
embryonic
phases ",
Extensive
chromosomal
chimeras were observed in callus derived regenerants
of tobacco
and transmission
of chromosomal
chimeras was reported to first and second selfed
progeny plants36.
For mutant selection, tobacco cells are a favourite
material due to the regenerative ability coupled with
the capability of tobacco callus to form fine cell
suspensions
that grow rapidly. Carlsorr" isolated
auxotrophic
mutants of tobacco using selection
technique developed for animal cell culture. The
nucleotide 5' brornodeoxyuridine was used to select
six tobacco clones showing partial growth requirements ". Clones were selected in N. plutnbaginifolia
requiring isoleucine, leucine and uracil38. Temperature sensitive variants of N. tabacutn were also
isolated successfully'". Of the specific selection
INDIAN J BIOTECHNOL,
174
agents, chlorate
has been found to be the most
effective with plant cells. Nitrate reductase converted
chlorite that kills plant cells. The cells lacking nitrate
reductase survived and could be isolated. This system
was so effective that Muller and Grafe"
isolated
double recessive mutants in N. tabacum.
Like auxotrophic
mutants, there are many reports
wherein
tobacco
cell lines resistant
to various
41
compounds
have been isolated. Widhlom
utilized
tryptophan analogue,S
methyltryptophan
to select N.
tabacum resistant
cell
lines.
In another
case,
suspension
cultures
resistant
to ethionine
were
isolated'",
Selection
for
resistance
to several
antibiotics, initially with the streptomycin,
produced
43
organelle encoded mutations in Nicotiana .
Tobacco cell lines tolerant to high NaCI levels
(0.88%)
have been isolated":".
The regenerated
plants from these line also retained tolerance through
two successive sexual generations
with the enhanced
46
levels of tolerance.
Dix
obtained
NaCI tolerant
plants in N. syLvestris and observed that the character
was transmitted to the next generation.
Different cell
lines of N. tabacum resistant to various herbicides,
such
as
Arnitrole'",
Bentazone'",
Paraquat",
Picloram5o
have
also
been
isolated.
Moreover,
glycerol and lactose were successfully
utilized for
detecting mutant cells of tobacco".
Other application
of mutant selection
includes
selection for disease resistance.
Plant resistance to
virus and its transmission
to the progeny of tobacco
has been reported.". Single clones of tobacco tissue
varied in their susceptibility
to virus and fungal
infections
and their multiplications.
Population
of
rnutagenized haploid cells of tobacco was plated in a
medium
containing
inhibitory
concentrations
of
methionine
since
Pseudomonas
tabacii, causal
bacteria of wild fire disease, produces methionine. In
vitro survived cells, produced on inhibitory levels of
methionine,
were further used for generating disease
resistant varieties ". In callus derived plants of an
interspecific
hybrid of tobacco, variability for plant
height, number of days to flower, number of leaves
and leaf area were also reported.".
Studies on
Protoplasts
Isolation,
Culture
and
Fusion
of
Isolation, culture, fusion of plant protoplasts
and
regeneration
has generated
great hopes for plant
improvement.
Absence of cell wall allows the fusion
between protoplasts, derived from two diverse plants
that are sexually incompatible,
as well as uptake of a
APRIL 2004
foreign genetic material, such as organelle or DNA,
into genome. For the first time, Nagata and Takebe55
descri bed the regeneration
of whole plants from
mesophyll protoplasts
of N. tabacum. Later, others
also reported
regeneration
of entire plants from
mesophyll
protoplasts
of tobacc056.57.
Protoplasts
derived from haploid tobacco have also shown the
regeneration
potential ". Leaves are considered
an
ideal source of protoplasts for tobacco because they
can be kept in abundant supply by ill vitro shoot tip
culture or from greenhouse
grown plants. Thus, a
large quantity of mesophyll protoplasts of tobacco can
be isolated from diploid or haploid plants. Moreover,
high percentage
of the protoplasts
can reform cell
wall and divide to produce a callus. Protoplasts have
also been isolated from epidermis, cell suspensions
and stem callus, beside mesophyll
cells. Further,
parameters
affecting
the initiation
of protoplast
division of haploid and diploid tobacco have been
evaluated ".
Somatic
hybridization
has
assumed
great
significance
among many applications
of protoplast
technology. The experiment to select somatic hybrids
with the aid of albino mutant was first performed on
tobacco haploids'". Similar results were obtained with
the interspecific
combinations
of non-allelic
light
sensitive albino mutants of N. sylvestris": Non-allelic
albino mutants were also used for N. tabacum and N.
knightiana, which was unable to produce shoots
forming
potential
of tobacco
and
chlorophyll
synthesis of N. knghtiana. The first somatic hybrids
plant was reported after a fusion of N. glauca x N.
langsdorffii
protoplasts
with the help of sodium
nitrate'". Protoplasts
from cell line of N. sylvestris
resistant to kanamycin
that have no ability to form
shoots were fused with the protoplasts
from N.
knightiana that do not form shoots in vitro. Somatic
hybrids were obtained on the basis of kanamycin
.
caoaci
h
63- .
resistance
an d on t hee capacity
to regenerate soots
Vigorous growth patterns of hybrid colonies were also
used to isolate hybrids from N. glauca and N.
langsdorfii". Several fusion experiments on tobacco
was conducted using plastorne mutants in relation to
chlorophyll
synthesis and cytoplasmic
male sterility
(CMS) and segregation
of mixed cytoplasm
into
mutant and wild type plastorne were observed65,66.
Gamma irradiated protoplasts, which were carrying a
functional nptII gene, were fused with un-irradiated
kanamycin-sensitive
recipient
protoplasts
to yield
asymmetric hybrids 67.
GANAPATHI
et al: TOBACCO FOR TISSUE CULTURE INTERVENTIONS
The somatic hybrid plants generally have chromosome number more than 4n due to the fusion of more
than one protoplast. The hybrid nature of most of the
somatic fusion products could be demonstrated by
their chromosome analysis, isoenzymes, morphological comparisons or growth characteristics. Prat68
examined mutations arised following protoplasts
culture of highly inbred line of N. sylvestris and also a
line derived from it after five consecutive cycles of
androgenesis and chromosome doubling. A system
was devised in N. tabacuni" and N. sylvestri/o where
somatic embryos were directly produced from
cultured protoplasts without callus formation. The
feasibility of this technique at the application level
will further determine the practicability of somatic
hybrids as to be complementary to classical plant
breeding
methods.
Besides
protoplast
fusion,
extensive experiments have been conducted on
tobacco protoplasts for various other aspects of
genetic manipulation.
Studies on Secondary Metabolites
formation
and Biotrans-
Plant
cell
cultures
synthesize
secondary
metabolites (biochemical totipotency) and this has
significance not only for basic research but also for
industrial processes. Ohta and Yatazawa" reviewed
the work on nicotine production in tobacco tissue
cultures. Nicotine in the range of 0.1-1 mg/mg dry wt
was detected in cell cultures of tobacco; whereas, 29
mg/mg of dry wt was found in the roots of intact
plants 72. It was observed that nicotine synthesis in
tobacco could be regulated by exogenous supply
without recourse to organogenesis". A close relationship has also been demonstrated between cell organization and nicotine production in tissue cultures of
tobacco". By obtaining single cell clones, high
yielding strains of nicotine were isolated. The
technique of single cell plating was used and a
number of cell colonies were isolated from cell
cultures of N. cellrustica, which showed wide
variations in their growth characteristics and ability to
synthesize nicotine". Further, the nicotine content in
tobacco showed relationship with the ploidy level of
the planr'". Besides nicotine, a number of other
secondary
products, such scopoletin,
esculetin,
bergapten, cycloartenol, citrostradienol, citroastradiol,
cycloeulenol, obtusifoliol " and aliphatic alkanes 77,
have been detected in tobacco cell cultures. Nicotiana
cell cultures also have the ability to transform organic
175
compounds and referred to as biotransformation, for
example stereospecific reduction of codeine." and
conversion of N-diphenylurea
into Dvglucose".
Microsomes from tobacco tissue cultures were also
found to convert squalene 2, 3 epoxide into cycloartenol'". These examples opened up the possibilities
of identifying a high yielding nicotine cell line. The
work on biotransformation in Nicotiana pertaining to
several compounds has shown the possibilities of
utilizing plant tissue and cell cultures for the isolation
of several useful compounds.
Studies on Transgenic Tobacco and Applications
Studies undertaken by Uchimiya and Murashige
with tobacco DNA and tobacco protoplasts showed
uptake of homologous DNA81. Suzuki and Takebe
demonstrated the insertion of viral DNA into
mesophyll protoplasts of tobacco'", Agrobacterium
tumefaciens, a soil bacterium, has been known to
induce crown gall disease in many plants. During
infection process, the bacterial plasmid integrates into
plant genome and influences the plant tissue to form
galls. Using A. tumefaciens, tobacco cell suspensions
were transforrnedv''" and showed the presence of
nopaline, the plasmid DNA encoding amino acid. The
initial experiments with tobacco generated interest in
the use of A. tumefaciens system for transformation
and, in recent years, its plasmid has become an
important vector for gene transfer. Venkateswarlu and
Nazar85 presented evidence by using tobacco
chloroplasts that Agrobacterium-mediated
transformation could be used to introduce foreign genes into
higher plant chloroplasts by site-specific homologous
recombination.
An unusual approach to transfer genes was
demonstrated in Nicotiana using irradiated pollen'".
Seeds were produced in N. forgetiana by pollinating it
with irradiated pollen of N. alta. Although, the
irradiated pollen produced pollen tube but failed to
fertilize the ovule. However, most of the plants
produced showed flower colour and other characters
of N. alta. In another approach, it has been shown that
swelling of germinating pollen grains could take up
DNA or bacteriophage", The progenies of N. glauca,
derived from N. glauca pollen treated with N.
langsdorffi DNA, produced tumours. The tobacco
protoplasts could also be transformed with disarmed
Ti plasmid vector pG 3850. Further, PN CA T
containing a chimeric PNOS CAT gene construct
gave rise to chloroamphenicol resistant calli.
176
INDIAN J BIOTECHNOL,
APRIL 2004
A simple and general method for transformation,
using tobacco leaf disks, was developed", which has
become the standard method for producing transgenic
tobacco. Leaf disks (1 cnr') were excised from in vitro
shoot cultures for preculture on MS medium with BA
and NAA. After 2 days, the leaf disks were cocultured with overnight grown A. tumefaciens culture.
After 30 min of co-infection, the leaf disks were
blotted to remove excess bacteria and transferred to
fresh culture plates of MS medium with BA and
NAA. Following three days of co-cultivation, the leaf
disks were transferred to selection medium with
kanamycin and cefotaxime or carbenicellin. The
regenerated shoots were rooted on rooting medium
containing NAA with high levels of kanamycin,
usually 100 mg/I. Those plants that rooted on this
medium were mostly considered to be transgenic and
could be studied further. Fig. 1 shows different stages
in the leaf disk transformation of tobacco var. Havana
425. A wide range of selectable markers are employed
in transformation experiments, however, kanamycin
has been the most extensively used.
Table 1 presents different transformation methods
used for the first time in tobacco89-94. Employing
tobacco as the experimental system, transfer of useful
genes have also been made, for example insect
resistance, herbicide tolerance, stress and disease
resistance.
For developing
herbicide
tolerance,
Arabidopsis
csr-J-J gene was transferred into
Canadian flue-cured variety, Delgold95. Resistance to
wildfire disease was introduced into transgenic
tobacco plants by expressing toxin resistance gene".
Very significant resistance to tobacco mosaic virus
has also been obtained by the expression of viral
replicase", Genetically modified, virus (CMV, TMV)
resistant tobacco yielded an average 5-7% more
leaves for processing and saved 2-3 applications of
insecticide applications. Virus resistant tobacco has
also been field tested in China since 1991 and is now
being used in industrial manufacturing for national
consumption. Using the codon-optimized S-endotoxin
gene from Bacillus thuringiensis, under the control of
CaMV 35S promoter, four lines were developed
based on the toxicity to tobacco horn worms in
greenhouse trials'", By expressing E. coli mannitol
dehydrogenase gene, increased mannitol accumulation in tobacco has been demonstrated".
Transgenic tobacco, expressing satellite RNA and
coat protein gene from cucumber mosaic virus
(CMV), showed resistance to CMV under both
Tobacco leatpieces were infectedwi1h
Agrobacterium (5 X 10' cells/mlJ and cocultivated on MS + SAP (1 mgAt) + NAA
(0.1 mg/lt). Alter 3 days the same were'
transterred to sam e media containing
CEtotaxime (400 mglltJ
Transterto MS + SAP (1 mglltJ + NAA
(0.1 mgAt) + Cet<ltaxim e (400 m9'lt) +
Kanamycin (50 m g'ItJ in pe!riplates
Transterto MS + SAP (1 mgllt) + NAA
(0.1 mgllt) + Cet<ltaxlme(400mgllt)+
Kanamycin (100 mgllt) in tubes
Transterto
MS + CEt<ltaxim e (400 mgllt) +
Kanamycin (1OOmgllt)
h"'''''''
.[],
Transter to green house for
hardening
TOBACCO TRANSFORMAllON
Fig. I-Different
stages in the Agrobacterium-mediated
transformation of tobacco var. Havana 425
Table I-Genetic
transformation
leaf disk
methods used in tobacco
S.No.
Method
Reference
I
Direct DNA uptake
Paszokowski et aZ89
2
Agrobacterium
Horsch et aZ90
3
Electroporation
Shillito et aZ91
4
Liposome mediated
Deshayes et aZ92
5
Microinjection
Crossway et aZ93
6
Particle bombardment
Klein et aZ94
mediated
natural and mechanical inoculations. Potato virus Y
coat protein gene was cloned and sequenced from an
Indian isolate'?". The coat protein gene was further
sub-cloned into plant expression vector pBINPLUS
and leaf discs of tobacco var. Havana 425 were
transformed using A. tumefaciens. Molecular analysis
confirmed the integration of the PV'Y' coat protein
gene. All the transgenic plants were morphologically
similar to non-transformed controls with respect to
appearance, flowering and seed set. The resistance
was assayed by infecting the control and transgenic
GANAPATHl
et at: TOBACCO FOR TISSUE CULTURE INTERVENTIONS
plants with the virus and the virus accumulation was
analysed through ELISA. All the transgenic lines
showed good amount of resistance against PV'Y'
accumulation
upon infection. The data clearly
demonstrate that the distinct Indian isolate of PV'Y'
can be used in imparting coat protein gene-mediated
resistance in tobacco and the level of resistance is not
correlated to the level of expression of the PV'Y' coat
protein in transgenic tobacco'l".
A synthetic substitution analogue of magainin,
MSI-99 was expressed in tobacco'?'. Magainin is one
of the earliest reported anti-microbial peptide isolated
from skin secretions of the African clawed frog,
Xenopus laevis. MSI-99 was sub-cloned into plant
expression vector pMS1168, wherein the peptide was
targeted to extracellular spaces. Tobacco plants
transformed
with pMSI168
showed
enhanced
resistance against Sclerotinia sclerotiorum, Alternaria
alternata and Botrytis cine ria pathogens. Tobacco
leaves (control and transgenic) were infected with
actively growing fungal mycelia of these pathogens
and lesion diameter was measured after three days of
infection. The leaves of transgenic tobacco plants
showed the significant reduction in lesion diameter
compared to control leaves, indicating the usefulness
of this peptide in enhancing the disease resistance in
tobacco plants.
Rol C gene was transferred into tobacco using A.
rhizogenes and transgenic clones for flowering, leaf
and flower size and height were isolated 102. As a
result, such transfer system is suggested for
modifying horticulture crops. However, rolA gene
was reported to stimulate nicotine production
directly 103. A cDNA and corresponding promoter
region, for a naturally occurring feedback insensitive
anthranilate synthase a subunit gene, has been
isolated from unselected but 5 methyl tryptophan
resistant tobacco cell line'?'. In an another experiment, the tobacco rdcS promoter fused to the GUS
reporter gene was delivered to black spruce via
microprojectile DNA bombardment and its regulation
was studied. The results showed that tissue specific
regulation of the rbcs promoter might be conserved
between tobacco and black sprucel05. The DNA
binding domain of yeast transcriptional activator
'(GAL 4) was demonstrated expressing in the
transgenic tobacco plants in order to attempt specific
repression of reporter genesl06. Transgenic tobacco
plants were obtained from tissues infected either with
a disarmed or a virulent root forming Agrobacterium
177
containing a binary vector':". Transgenic tobacco
plants were also achieved through microtargeting'l".
Further, the cells of Escherechia coli and A.
tumefaciens were used as microprojectiles to deli ver
DNA into suspension cultured tobacco cells, using
helium powdered biolistic device, and obtained
hundreds of transient transformants per bombardment
but did not get any stable transformants 109.
Phytoremediation, i.e. bioremediation using plants,
is becoming a reality for safe removal of organic
compounds from contaminated water and soil. The
natural ability of certain plant species to take up
heavy metals and radioactive elements is being
exploited for bioremediation
efforts. Transgenic
tobacco plants, genetically engineered to express a
bacterial enzyme, were developed to detoxify TNT
(2,4-6 trinitrotoluene) 110.A wide variety of naturally
occurring soil bacteria possess a wide array of
enzymes capable of degrading insecticides and
herbicides. Genes encoding such enzymes can be
cloned and expressed in transgenic tobacco plants for
use in phytoremediation.
Tobacco plant has been extensively used for
transgenic research and continues to remain as the
model plant of choice 1". However, as the development proceeded in the area of genetic transformation,
Arabidopsis and other economically important plants
(rice) became the other choice. The first field trial of
transgenic crops was conducted with tobacco in
France and USA in 1986. Tobacco was also the first
crop to be commercialized in China and USA in 1991
and 1994, respectively. Transgenic plants resistant to
TMV were grown in almost 1 million ha in China in
1994, which yielded 5-7% more leaves'V.
Studies on Molecular Farming
Several recombinant proteins are being produced in
transgenic plants as bioreactors for the large-scale
production of commercially important compounds
of pharmaceutical
and
industrial
importance
(Table 2)"3-118. Tobacco has served as the choice
plant system for the production of potential
therapeutic proteins in plants 1119,120.
Tobacco is an
ideal plant-bioreactor for molecular farming because
the pharmaceutical protein is produced in its leaves
(about 40 tons of leaves/acre) and the production
system does not require flowering. The leaves contain
10% protein and about 2000 kg proteinlacre/yr can be
produced. Tobacco is also a prolific seed producer
with about 1 million seeds/plant. The first transgenic
INDIAN J BIOTECHNOL,
178
Table 2-Production
of biopharmaceuticals
APRIL 2004
for human health in transgenic tobacco plants
Protein
Application
Expression level
Reference
Human Protein C
Anticoagulant
0.01% SLP
Crammer
Human granulocyte macrophage calony
Neutropenia
Giddings
lll
et al
l14
et al
stimulating factor
Human somatotropin
Growth hormone
7% SLP (chloroplasts)
Staub
Human erythropoietin
Anemia
0.01%SLP
Kusnadi
Human epidermal growth factor
Wound repair and cell proliferation
0.01%SLP
Crammer
et al
et all16
et alliS
l16
et al
ill
Human interferon beta
Hepatitis Band C
0.01% FW
Kusnadi
Human haemoglobin alpha, beta
Blood substitute
0.05% SP
Crammer &
Weissenborn
0.01%FW
Ruggiero
et alliS
Giddings
et al
Human homotrimeric collagen
Collagen
Angiotensin converting enzyme
Hypertension
Alpha Trichosanthin
from TMY-U1
I 17
l14
HIY therapy
2% SLP
Giddings
et all14
Gauchers disease
1-10% SLP
Crammer
et al
subgenomic coat protein
G Iucocerebrosi dase
11l
FW: fresh weight, SLP: soluble leaf protein, SP: seed protein, -: not reported
plant synthesized product is a tobacco derived
antibody targeting gum disease. Most of the tobacco
types secrete sticky compounds on the outer leaf
surface as a first line of defence and some varieties
produces 16% of the leaf dry weight as gum.
Scientists of the University of Kentucky isolated a
promoter to control the type of compounds that are
secreted by tobacco leaf hair. Targeting the
recombinant proteins in tobacco gum offered several
advantages and purification would be easier and much
cheaper!".
Various types of antibodies are used in human
medicine for diagnostic as well as for therapeutic
purposes 122. Recombinant
antibodies have been
produced in transgenic tobacco plants. The expression
and assembly of immunoglobulin (lgG heavy and
light chains) led to the production of other antibodies,
like IgG-IgA antibody against a surface antigen of
Sterptococcus mutans designed to prevent tooth
decay. Estimates of production costs for an antibody
in plants indicate as much as 10 to 20-fold lower costs
per gram compared to produced by cell culture. Since
plant based expression levels are low, chloroplast
transformation may offer as an alternative. As there
are 10,000 copies of chloroplast genomes per cell, this
can facilitate the introduction of 10,000 copies of
foreign genes per transformed cell and subsequently
can boost several hundred-fold increase in gene
expression compared to nuclear transformation 123.
Vaccines are of prime significance for the human
health. However, in many developing countries, the
incidence of infectious diseases and the expense of
immunization programme limit the use of available
vaccines for large segments of population. Vaccine/
antigen coding genes for traveller's diarrhoea,
hepatitis B, gastroenteritis, foot and mouth disease,
mink enteritis, swine fever, hog cholera, rabies, swine
transmissible gastroenteritis,
dental caries, autoimmune diabetes and cholera have been expressed in
different plant systems including tobacco (Table 3)124.
137. The first study on the production of plant-based
vaccine was done in tobacco with the hope of
developing a less expensive product 130. It was also
shown that rHBsAg self-assembled into sub viral
particles, identical to the plasma and yeast derived
HBsAg specific antibodies in mice122. The expression
of cholera toxin B was demonstrated in transgenic
tobacco chloroplast, resulting in the accumulation of
4.1 % of total soluble leaf protein as functional CTB
oligomers'i". Further, binding assays confirmed the
correct folding and disulfide bond formation of the
plant derived CTB pentamers. The expression of Cterminal
region
of merozite
surface
protein
(PfMSP119), a potential malaria vaccine candidate,
was reported in tobacco 128. Immunoblot
assay
indicated that transformed plant expressed MSP119
displayed structural and immunological
characteristics identical to the E. coli expressed protein. This
presents a significant step towards the development of
low-cost subunit vaccine against malaria.
.
The tobacco cell line (NT -1) was transformed with
hepatitis B virus's'
gene, coding for surface
GANAPATHI
et 01: TOBACCO FOR TISSUE CULTURE INTERVENTIONS
Table 3-Vaccines
produced in transgenic tobacco plants
Disease/Antigen
Origin
Traveller's
Enterotoxigenic
Diarrhoea
179
E. coli
Expression level
Reference
0.001% SLP
Haq et 01124
(Heat labile enterotoxin B)
01125
Hepatitis B (HBsAg)
Hepatitis B virus
0.0066% SLP
Mason
Gastroenteritis
Norwalk virus
0.23% SLP
Mason et 01125
et
(Norwalk virus capsid protein)
Immunocontraception
(ZP 3)
Fitchen et 01126
Murine
Turpen et aim
Malaria (epitopes derived
Plasmodium sporozoites
from sporozoites)
P lasmodi um falciparum
Ghosh et ai128
Swine fever (Hog cholera, EO, El & E2)
Swine fever virus
Kapusta et
Dental caries (SpA antigen)
Streptococcus inutans
Cholera(CT-B)
Vibrio cholerae
0.0035% SLP
0.02% SLP
130
Verch et ai132
Beachy et ail33
Influenza virus
Lymphoma (Tumour derived ScFv epitopes)
Post--surgical/burn infections
Mason & Arntzen
Arakawa et ai131
Colon cancer
Influenza
ctf129.
Mc Cormick et
134
ctf
,
135
Stackzek et ai
Pseudomonas aeruginosa
Epitope of outer membrane protein F
Human cytomegalo virus
Cytomegalo virus
0.02% SLP
Tackaberry et 01136
Swine Transmissible gastroenteric virus
Corona virus
0.20% SLP
Tuboly et ai137
SLP: soluble leaf protein, -: not reported
•
138
TIC
.
antigen':".
_wo pant
transrorrnanon
vectors
pHERlOO and pHBSlOO with and without endoplasmic reticulum retention signal, respectively were
used for transformation. The integration of the
transgene was analysed by PCR and southern blot
hybridization, and expression level was determined by
ELISA. The maximum expression of 2 ug/g fresh
weight and 10 ng/ml of spent medium was reported in
pHERI00 transformed cells. Western blot analysis
confirmed the presence of 24 kDa band specific to
HBsAg in the transformed cells. The buoyant density
in CsCI of HBsAg deri ved from pHBS 100
transformed tobacco cells was determined and found
to be l.095 gm/mJ. The secretion of HBsAg particles
by plant cells into the cell culture medium was
reported for the first time.
The availability of genetic transformation methods
of. plants has broadened the type of experimental
problems, the diversity of cell types that can be
approached and the transfer of useful genes. These
investigations promise to make the plant based
systems extremely attractive and powerful for crop
improvement, besides studying gene regulation and
developmental biology in higher plants.
Conclusions
Tobacco has become the plant system of choice for
almost all the aspects of cell and tissue culture
research. Majority of the experimental discoveries in
the field of plant cell, tissue culture and plant
molecular biology owe their inception to the studies
with tobacco. Trends in plant biotechnology research
show a substantial increase in research publications
on tobacco from 123 to 1396 from 1980-1990 to
1990-2000. Next to tobacco, arabidopsis has been
now the model plant for molecular research.
However, researchers all over the world still continue
to use tobacco for a wide variety of plant research
programmes. Tobacco as a model system has played a
major role in the advancement of plant science and
has been used as a tool in gaining fundamental
knowledge in diverse areas of plant biology. Recent
advances in the field of molecular farming have used
tobacco as a 'plant factory' for the purposes of
developing
production
system for recombinant
proteins,
pharmaceuticals,
vaccines,
industrial
enzymes and antibodies. Several companies are
already using this technology for commercial
production. Vector Tobacco Inc., Durham, USA has
developed a variety of genetically engineered tobacco
INDIAN J BIOTECHNOL,
180
plants. One of them is nearly nicotine-free. Based on
the present developments, it appears that, in future,
tobacco being a non-food crop and having apathy
from non-cigarette lovers, will assume the role of a
most useful crop for molecular farming, leading to
better industrial and human healthcare options.
18
19
20
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